Flora 212 (2015) 30–37 Contents lists available at ScienceDirect Flora j ournal homepage: www.elsevier.com/locate/flora Eco-physiological adaptations of Panicum antidotale to hyperosmotic salinity: Water and ion relations and anti-oxidant feedback a b c d,∗ Tabassum Hussain , Hans-Werner Koyro , Bernhard Huchzermeyer , M. Ajmal Khan a Institute of Sustainable Halophyte Utilization (ISHU), University of Karachi, Karachi 75270, Pakistan b Institute of Plant Ecology, Justus-Liebig University Gießen, Heinrich-Buff-Ring 26–32, D-35392 Gießen, Germany c Institute of Botany, Leibniz University Hannover, Herrenhäuser Str. 2, D-30419 Hannover, Germany d Centre for Sustainable Development, College of Arts and Sciences, Qatar University, Doha, Qatar a r t i c l e i n f o a b s t r a c t Article history: Threshold of salt resistance of plants is determined by their response to osmotic and ionic stress (pri- Received 28 July 2014 mary constraints) imposed upon them. However, recent reports emphasize the importance of secondary Received in revised form 24 January 2015 constraints like oxidative stress. The aim of this study was to determine the effect of salinity on growth, Accepted 16 February 2015 mineral nutrition, water relations, compatible solutes, and the antioxidant system in Panicum antidotale. Edited by Hermann Heilmeier. Five levels of salinity (0, 125, 250, 375 and 500 mM NaCl) were applied using a quick check system in a Available online 18 February 2015 fully randomized greenhouse study. Plant growth parameters, water relations, organic (proline and solu- + + ++ ++ ble sugars), inorganic osmolytes (Na , K , Ca and Mg ), and macronutrients such as carbon or nitrogen Keywords: were measured beside the activities of the antioxidant enzymes superoxide dismutase (SOD), cata- Salt resistance lase (CAT), ascorbate peroxidase (APx) and glutathione reductase (GR) and non-enzymatic antioxidant Water relations Osmolyte metabolites (oxidized and reduced ascorbate). Oxidative stress Sodium chloride concentrations above 125 mM substantially inhibited growth. This inhibition was Antioxidant enzymes attributed to high energy costs needed for osmotic adjustment, ion compartmentalization, synthesis of organic osmolytes (such as proline and sugars), ROS scavenging and the maintenance of ionic homeostasis. The plants resisted against oxidative stress by increasing activities of antioxidant enzymes such as SOD, APx, GR and CAT and elevating levels of oxidized and reduced ascorbate (DAsA and AsA) at higher salinity. They also maintained a low redox ratio of ascorbate/dehydro-ascorbate and therefore a high capacity to manage oxidative stress. Thus P. antidotale is capable of managing ROS stress at high salinity and therefore can be sustainably grown as a fodder crop in saline arid regions. © 2015 Elsevier GmbH. All rights reserved. −1 −1 Introduction and/or waste land. The plant produces about 60 t ha year fresh biomass on saline soils with brackish water irrigation and can be Soil salinity is one of the most threatening abiotic factors espe- used to replace maize as cattle feed (Khan et al., 2009). However, cially for arid and semiarid regions where it decreases conventional saline water irrigation requires precision, and accurate knowledge agricultural productivity (Gleick et al., 2011) through reducing about sustainable growth conditions needs to be assured. Therefore growth and survival of plants (Shi and Wang, 2005; Sobhanian et al., this study develops arguments regarding underlying mechanisms 2010). Halophytes have adapted to counter harmful effects of soil of salt resistance of Panicum (Koyro et al., 2013) up to levels of salinity and are able to complete their life cycle in saline habitats seawater salinity. (Flowers and Colmer, 2008; Munns et al., 2010). The utilization of Hyperosmotic salinity limits plant growth primarily by low salt resistant plants as non-conventional alternate sources of food, water availability (osmotic effect) and high salt concentrations fodder, wood, medicine, oil, biofuel and industrial raw material, and (ionic effect) (Munns and Tester, 2008). The scarcity of water can for land reclamation and ornamental purposes is well established reduce leaf cell expansion and stomatal conductance and is fre- (Abideen et al., 2012; Khan et al., 2009; Qasim et al., 2010). Panicum quently accompanied by a reduced CO2 fixation. Latter one can antidotale has a considerable agrarian potential to be used as cattle contribute to oxidative stress (Koyro et al., 2013). Halophytes + feed and it can be grown with saline water irrigation of salinized reduce the osmotic potential by absorbing Na and they avoid its + toxicity by compartmentalizing Na in the vacuole (Lee et al., 2007). + High concentrations of Na in the medium may alter the transport + + ∗ and uptake of K (Marcum, 2008; Zhou and Yu, 2009) leading to K Corresponding author. Tel.: +974 4403 4953; fax: +974 4403 4931. + + E-mail address: [email protected] (M.A. Khan). starvation and/or [K ]/[Na ] imbalance (Ahmed et al., 2013; Hauser http://dx.doi.org/10.1016/j.flora.2015.02.006 0367-2530/© 2015 Elsevier GmbH. All rights reserved. T. Hussain et al. / Flora 212 (2015) 30–37 31 + + ◦ and Horie, 2010). However, Na toxicity and/or K deficiency in the measured. Plants were dried in an oven at 70 C until constant cytoplasm and its organelles (ionic effect) may reduce or inhibit weight was obtained. plant growth (Sobhanian et al., 2011) and can be an additional reason for oxidative stress. Down-regulation to low cytoplasmic Water relations water potential, ionic homeostasis and the protection of proteins, protein complexes and membranes against oxidative stress are fre- Water potential was measured on intact leaves by the dew point quently ensured for instance by the accumulation of compatible method with HR-33 T Dew Point Microvolt meter, using a L-51- organic solutes, e.g. amino acids (proline and citrulline), onium SF leaf chamber (Wescor, USA). Relative water content (RWC) was compounds (glycinebetaine, 3-dimethylsulfonoproprionate), car- measured using 3 discs of 1 cm diameter from a leaf (avoiding mar- bohydrates and polyols (Subudhi and Baisakh, 2011; Verbruggen gins and midrib) and fresh weight (FW) was determined (Sharp and Hermans, 2008). Some of these compatible osmotic substances et al., 1990). Discs were placed in 1.5 ml of deionized water for 4 h such as proline act as ROS scavenger because they use protons dur- ◦ at 4 C to let the tissue absorb water. Thereafter weight of the discs ing their synthesis and therefore help in reducing oxidative stress was termed as turgid fresh weight (TFW). Tissues were dried at (Kocsy et al., 2013). The reduction of CO assimilation via ionic and 2 ◦ 70 C for 48 h and dry weight (DW) was determined. RWC (%) was osmotic effects can disturb this balance due to over-reduction of calculated as: the photosynthetic electron transport chain. Consequently produc- tion of active oxygen species (ROS) such as superoxide or hydrogen FW − DW = peroxide may be stimulated (Koyro et al., 2013). RWC (%) × 100 TFW − DW ROS have a high affinity to react with proteins, lipids and nucleic acids and cause the malfunctioning of these macromolecules (Kocsy et al., 2013). However, several antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidases Proline and sugar determination (APx) and glutathione reductase (GR) along with non-enzymatic ROS scavengers like nitrate, ascorbate, glutathione, carotenoids and Proline was estimated by using powdered plant material tocopherols can detoxify ROS (Jithesh et al., 2006). A strong antiox- (50 mg) which was mixed with 4 ml of 3% sulphosalicylic acid and ◦ idant response mechanism is of vital significance for plants coping sonicated at 30 C for 15 min and then centrifuged at 3000 × g. This with low soil water potential, hyperosmotic salinity and nutrient extract was used for measuring proline content. One ml acid nin- imbalance (Jithesh et al., 2006). It was shown for instance that the hydrin and 1 ml of glacial acetic acid were added to the reaction ◦ excessive production of ROS in P. antidotale at high salinity can lead mixture which was boiled at 100 C for 1 h. The reaction was termi- to an increased lipid peroxidation of membranes causing leakage nated in ice and 2 ml of toluene were added to the reaction mixture (Koyro et al., 2013). which was vortexed for exactly 30 s. The absorbance of the upper The aim of this study was to determine the effect of salinity on phase was measured at 520 nm and proline concentration was esti- growth, mineral nutrition, water relations, compatible solutes, and mated against a standard curve using l-proline (Bates et al., 1973). the antioxidant system in P. antidotale. These information will be Soluble sugars were determined by the anthrone method (Ludwig used to analyze interactive responses on salinity constraints. and Goldberg, 1956). Press sap was used for measuring sugars. Samples were heated for 11 min with anthrone reagent (in 95% H SO ) and the reaction was terminated in ice. The absorbance was Materials and methods 2 4 recorded at 630 nm and sugars were estimated by using sucrose as a standard. Plant material and treatments Surface sterilized seeds of Panicum antidotale (wild type) were Cations, carbon and nitrogen analysis germinated in soil type LD 80 (Fa. Archut, Vechta, Germany) in an environmentally controlled green house (Giessen, Germany). 200 mg of dried leaves (adult and juvenile), stem and root were After 2 weeks young seedlings were transplanted to a soil less ◦ ashed in an oven at 600 C for 8 h. The ash was dissolved in 20% (gravel/hydroponics) quick check system (Koyro 2006). The plants HNO3 on Bunsen burner at gentle flame. The mixture was filtered were irrigated with a basic nutrient solution as modified by Epstein + + ++ through ash-less filter paper (Whatman no. 40). Na , K , Ca and (1972) under 16 h light/8 h dark photoperiod for 3 more weeks. ++ ◦ ◦ Mg were measured with an atomic absorption spectrophotome- Temperatures were 27 ± 2 C during the day and 17 ± 2 C during ter (AAS 2100, PerkinElmer).